Interferon gamma (IFNγ) is a pleiotropic cytokine produced by T- and NK-cells and is involved in the regulation of both innate and adaptive immune responses. The major biological activities of IFNγ are associated with antiviral and immunomodulatory effects, cell grow and differentiation, and control of apoptosis (Stark, G., et al., Annu Rev Biochem, 1998, 67, 227-264; Ramana, C., et al., Trends Immunol, 2002, 23, 96-101; van Boxel-Dezaire, A., et al., Curr Top Microbiol Immunol, 2007, 316, 119-154). The IFNγ receptor is composed of two distinct subunits, IFNGR1 and IFNGR2, in which IFNGR1 is the major ligand-binding subunit, (Stark, G., et al., Annu Rev Biochem, 1998, 67, 227-264; Bach, E., et al., Annu Rev Immunol, 1997, 15, 563-591) while IFNGR2 plays a critical role in the generation of IFNγ signals (Hemmi, S., et al., Cell, 1994, 76, 803-810; Soh, J., et al., Cell, 1994, 76, 793-802). Interaction of IFNγ with its cell surface receptor activates receptor-associated Janus-activated kinase 1 (JAK1) and JAK2, which in turn phosphorylate and activate the signal transducer and activator of transcription-1α (STAT-1α). Phosphorylated STAT-1α dimerizes and translocates into the nucleus where it binds to well-defined DNA sequences called gamma interferon activation sites (GASs) in IFNγ-inducible promoters and activates the transcription of genes that encode members of the interferon regulatory factor (IRF) family of transcription factors (Stark, G., et al., Annu Rev Biochem, 1998, 67, 227-264; Boehm, U., et al., Annu Rev Immunol, 1997, 15, 749-795; Darnell, J. Jr., Science, 1997, 277, 1630-1635).
Ten members of the IRF family have been identified, including IRF-1, IRF-2, IRF-3, IRF-4/lymphoid-specific IRF/Pip/ICSAT, IRF-5, IRF-6, IRF-7, ICSBP/IRF-8, ISGF3γ/p48, and vIRF. IRF-1 and IRF-2 are the best-characterized members of this family (Nguyen, H., et al., Cytokine Growth Factor Rev, 1997, 8, 293-312; Miyamoto, M., et al., Cell, 1988, 54, 903-913; Harada, H., et al., Cell, 1989, 58, 729-739) and were initially identified by studies of transcriptional regulation of the IFN system. They have subsequently been shown to be key factors in the regulation of cell growth through their effects on the cell cycle (Taniguchi, T., et al., J Cancer Res Clin Oncol, 1995, 121, 516-520; Vaughan, P., et al., J Mol Med, 1997, 75, 348-359). IRF-1 is thought to function in a manner analogous to the tumor suppressor p53, activating a set of genes whose products are required for negative regulation of cell growth. IRF-2, which shares significant sequence similarity to IRF-1 within the DNA binding domain, represses IRF-1 regulatable genes (Taniguchi, T., J Cell Physiol, 1997, 173, 128-130). Although ICSBP/IRF-8 is thought to be expressed exclusively in cells of macrophage and lymphocyte lineage, (Driggers, P., et al., Proc Natl Acad Sci USA, 1990, 87, 3743-3747) it has been shown that the ICSBP gene is transcriptionally activated in human retinal pigment epithelium cell by IFNγ (Li, W., et al., Invest Ophthalmol Vis Sci, 1999, 40, 976-982).
In addition to JAK/STAT, IFNγ activates several other signal transduction proteins, including the mitogen-activated protein (MAP) kinases (Ramana, C., et al., Trends Immunol, 2002, 23, 96-101; van Boxel-Dezaire, A., et al., Curr Top Microbiol Immunol, 2007, 316, 119-154; Platanias, L., et al., Exp Hematol, 1999, 27, 1583-1592; Platanias, L., Nat Rev Immunol, 2005, 5, 375-386; Maher, S., et al., Curr Med Chem, 2007, 14, 1279-1289). MAP kinases are a superfamily of serine-threonine kinases that play important roles in various signal transduction pathways in mammalian cells. Three major MAP kinases have been identified—the extracellular signal—regulated kinase (ERK), the c-Jun NH2 terminal kinase (JNK), and the P38 MAP kinase (Chang, L., et al., Nature, 2001, 410, 37-40; Schaeffer, H., et al., Mol Cell Biol, 1999, 19, 2435-2444). Although emerging evidence exists that suggests a role for P38 MAP kinase in mediating fast cellular responses to IFN stimulation and in maintaining a more sustained response through regulation of gene transcription, (Platanias, L., Nat Rev Immunol, 2005, 5, 375-386; Chang, L., et al., Nature, 2001, 410, 37-40; Katsoulidis, E., et al., J Interferon Cytokine Res, 2005, 25, 749-756; Platanias, L., Pharmacol Ther, 2003, 98, 129-142; Li, Y., et al., J Biol Chem, 2004, 279, 970-979; Pearson, G., et al., Endocr Rev, 2001, 22,153-183) the precise function P38 MAP kinase in IFN signaling remains unclear (Platanias, L., et al., Exp Hematol, 1999, 27, 1583-1592). P38 MAP kinase may function as an IFNα- and IFNγ-dependent serine kinase for STAT, which is needed for STAT1 transcriptional activity (Goh, K., et al., Embo J, 1999, 18, 5601-5608). It also appears that P38 MAP kinase plays a role in the induction of antiviral responses (Goh, K., et al., Embo J, 1999, 18, 5601-5608). In RPE cells, P38 MAPK has been shown to be activated in response to oxidative stress, but no evidence exists that IFNγ can activate P38 MAPK (Faure, V., et al., J Biol Chem, 1999, 274, 4794-4800). Recently it has been shown that P38 MAPK mediates the flagellin-induced activation of CFTR-dependent Cl secretion in Calu-3 airway epithelial cells (Zhang, Z., et al., Infect Immun, 2007, 75, 5985-5992; Illek et al., 2008, in press).
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl channel located at the apical membrane of most epithelia. It helps to control transepithelial electrolyte transport, fluid flow, and the chemical composition of the extracellular spaces surrounding major organ systems such as intestine, sweat glands, lung and pancreas. CFTR belongs to the ATP binding cassette (ABC) superfamily of membrane proteins and has transmembrane domains (TMD1 and TMD), nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. The gating of CFTR is tightly regulated by protein phosphorylation and nucleotide concentration. It is now well-established that CFTR gene expression is regulated by many factors, including cytokines, in a cell- and stimulus-specific manner that can involve both transcriptional and posttranscriptional mechanisms (Kulka, M., et al., J Pharmacol Exp Ther, 2005, 315, 563-570).
As a lymphocyte effector molecule, IFNγ has been implicated in the pathogenesis of a number of intraocular inflammatory diseases of infectious or presumed autoimmune origin (Chiba, H., et al., Sci STKE, 2006, 2006, pe1; Fang, Y., et al., Thyroid, 2007, 17, 989-994; Willenborg, D., et al., J Neuroimmunol, 2007, 191, 16-25). In the eye, IFNγ plays important roles in macrophage activation and in the recruitment of inflammatory cells to sites of inflammation, and has been detected in vitreous aspirates of patients with uveitis, proliferative vitreoretinopathy, and other inflammatory eye diseases (Hooks, J., et al., Invest Ophthalmol Vis Sci, 1988, 29, 1444-1451; Limb, G., et al., Eye, 1991, 5(Pt 6), 686-693; Franks, W. et al., Curr Eye Res, 1992, 11 Suppl, 187-191; Ooi, K., et al., Clin Med Res, 2006, 4, 294-309). The retinal pigment epithelium (RPE), is a highly specialized derivative of the neuroectoderm with multiple roles in the maintenance of normal ocular function.
The retinal pigment epithelium (RPE) is single monolayer of epithelial cells, located in the back of the vertebrate eye, between the choroidal blood supply (choriocapillaris) and the neuroretina. The RPE acts as one of the components of the blood-retinal barrier, and RPE cells play vital roles in maintaining the visual cycle, in photoreceptor outer segment phagocytosis, and in transport of nutrients, metabolic waste products, ions, and fluid between the distal retina and the choriocapillaris. Dysfunction of RPE cells has been implicated in inflammatory and degenerative diseases of the retina and choroid, (Campochiaro, P., Expert Opin Biol Ther, 2004, 4, 1395-1402; Donoso, L., et al., Surv Ophthalmol, 2006, 51, 137-152; Voloboueva, L., et al., Invest Ophthalmol Vis Sci, 2005, 46, 4302-4310; Shi, G., et al., Invest Ophthalmol Vis Sci, 2008; Li, R., et al., Invest Ophthalmol Vis Sci, 2007, 48, 5722-5732; Jia, L., et al., Invest Ophthalmol Vis Sci, 2007, 48, 339-348) but relatively little is understood regarding the direct effects of inflammatory mediators on RPE physiology or pathophysiology.
A need thus exists in the art for elucidating whether IFNγ in plays a role in the dysfunction of RPE cells in order to develop methods for treating the numerous ocular diseases and disorders associated with RPE dysfunction.